Escherichia coli possesses two DNA glycosylase/apurinic lyase activities with overlapping substrate specificities, endonuclease III and endonuclease VIII, that recognize and remove oxidized pyrimidines from DNA. Endonuclease III is encoded by the nth gene. Endonuclease VIII has now been purified to apparent homogeneity, and the gene, nei, has been cloned by using reverse genetics. The gene nei is located at 16 min on the E. coli chromosome and encodes a 263-amino-acid protein which shows significant homology in the N-terminal and C-terminal regions to five bacterial Fpg proteins. A nei partial deletion replacement mutant was constructed, and deletion of nei was confirmed by genomic PCR, activity analysis, and Western blot analysis. nth nei double mutants were hypersensitive to ionizing radiation and hydrogen peroxide but not as sensitive as mutants devoid of base excision repair (xth nfo). Single nth mutants exhibited wild-type sensitivity to X rays, while nei mutants were consistently slightly more sensitive than the wild type. Double mutants lacking both endonucleases III and VIII exhibited a strong spontaneous mutator phenotype (about 20-fold) as determined by a rifampin forward mutation assay. In contrast to nth mutants, which showed a weak mutator phenotype, nei single mutants behaved as the wild type.Free radicals are produced in cells by ionizing radiation, a variety of chemical agents, and normal oxidative metabolism. The spectrum of free radical-induced damage to DNA is broad and includes a wide variety of modifications to the purine and pyrimidine bases, sites of base loss and single-strand breaks (for reviews, see references 11, 39, and 66). The 5,6 double bond of pyrimidines is particularly vulnerable to free radical attack (66), and for DNA thymine, the products include 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol [Tg]), 5-hydroxy-5,6-dihydrothymine, 6-hydroxy-5,6-dihydrothymine, and 5,6-dihydrothymine (produced under anaerobic conditions). 5-Hydroxymethyluracil (24) and a number of ring contraction and fragmentation products such as 5-hydroxy-5-methylhydantoin, methyltartronyl urea, and urea are also formed. A major product of free radical attack on cytosine is 5,6-dihydroxy-5,6-dihydrocytosine (cytosine glycol) (67), which is unstable and dehydrates to form 5-hydroxycytosine (5-OHC) or deaminates to form uracil glycol (Ug), which can also dehydrate to form 5-hydroxyuracil (5-OHU). Contraction and fragmentation products can also be found after free radical attack on DNA cytosine. Of the above-mentioned structures that retain intact ring conformation and base-pairing capabilities, only Tg has been shown to be a potentially lethal lesion. Tg is a potent block to DNA synthesis in vitro (17,28,31,56), using a variety of DNA polymerases, and when present in biologically active single-stranded phage DNA molecules is a lethal lesion with an inactivation efficiency of 1 (1, 29). Tg is also lethal in duplex X DNA, where it takes about 10 to 12 Tg lesions to kill (41). Other intact pyrimidine products su...
It has been postulated that ionizing radiation produces a unique form of cellular DNA damage called ''clustered damages'' or ''multiply damaged sites''. Here, we show that clustered DNA damages are indeed formed in Escherichia coli by ionizing radiation and are converted to lethal double-strand breaks during attempted baseexcision repair. In wild-type cells possessing the oxidative DNA glycosylases that cleave DNA at repairable single damages, doublestrand breaks are formed at radiation-induced clusters during postirradiation incubation and also in a dose-dependent fashion. E. coli mutants lacking these enzymes do not form double-strand breaks postirradiation and are substantially more radioresistant than wild-type cells. Furthermore, overproduction of one of the oxidative DNA glycosylases in mutant cells confers a radiosensitive phenotype and an increase in the number of double-strand breaks. Thus, the effect of the oxidative DNA glycosylases in potentiating DNA damage must be considered when estimating radiation risk.A pproximately 70% of radiation-induced DNA damages are formed by reactive-free radicals produced by the radiolysis of water in the vicinity of DNA (1). These radiation-induced DNA damages are repaired by base-excision repair (2) and overlap substantially with those formed during normal oxidative metabolism (3), which are produced at significant rates in unirradiated cells (4). This overlap of damages has led to a controversy with respect to determining radiation risk. Proponents of a ''threshold effect'' claim that because endogenous oxidative damages are effectively repaired and adaptive responses have been demonstrated for certain radiation endpoints, then a threshold must exist for the carcinogenic and lethal consequences of ionizing radiation (5, 6). This issue is an important one because much of the projected radiation exposures associated with human activity over the next hundred years will come from low doses associated with medical tests, waste cleanup, and materials associated with nuclear weapons and nuclear power. Whether a threshold exists for the consequences of radiation damage will significantly influence risk estimates for low-dose exposures. Currently, risk estimates for individuals or groups exposed to low radiation doses are determined from epidemiological data obtained from populations exposed to high doses, and a ''linear-no-threshold'' estimation is used for assessment. Proponents of the linear-no-threshold model hold that the biologically important radiation damages in DNA, such as double-strand breaks, are substantially different from single, repairable oxidative lesions. It also has been predicted that ionizing radiation produces a unique form of DNA damage called ''clustered damages '' (1, 7, 8). If, indeed, clustered damages can be demonstrated in living cells and if, unlike the single lesions from which they are formed, they are poorly repaired, these facts would provide additional support for the linear-nothreshold model.Modeling of radiation track structures (9, 10) ...
Energy from low LET ionising radiation, such as X rays and gamma rays, is deposited in the water surrounding the DNA molecule such that between 2 to 5 radical pairs are generated within a radius of I to 4 nm. As a result, multiple single lesions, including oxidised purine or pyrimidine bases, sites of base loss, and single-strand breaks, can be formed in DNA from the same radiation energy deposition event. The single lesions in these so-called multiply damaged sites or clustered lesions are repaired by base excision repair. Here we show that clustered DNA damages are formed in bacterial cells by ionising radiation and are converted to lethal double-strand breaks during attempted repair. In wild type cells possessing the oxidative DNA glycosylases that recognise and cleave DNA at repairable single damages, double-strand breaks are formed at radiation-induced clusters during post-irradiation incubation and in a dose-dependent fashion. Mutant cells lacking these enzymes do not form double-strand breaks post-irradiation and are substantially more radioresistant than wild type cells. These radioresistant mutant cells can be made radiosensitive by overexpressing one of the oxidative DNA glycosylases. Thus the effect of the oxidative DNA glycosylases in potentiating DNA damage must be considered when estimating radiation risk.
The Saccharomyces cerevisiae Cdc42p GTPase is localized to the plasma membrane and involved in signal transduction mechanisms controlling cell polarity. The mechanisms of action of the dominant negative cdc42 D118A mutant and the lethal, gain of function cdc42 G12V mutant were examined. Cdc42 D118A,C188S p and its guanine-nucleotide exchange factor Cdc24p displayed a temperature-dependent interaction in the twohybrid system, which correlated with the temperature dependence of the cdc42 D118A phenotype and supported a Cdc24p sequestration model for the mechanism of cdc42 D118A action. Five cdc42 mutations were isolated that led to decreased interactions with Cdc24p. The isolation of one mutation (V44A) correlated with the observations that the T35A effector domain mutation could interfere with Cdc42 D118A,C188S p-Cdc24p interactions and could suppress the cdc42 D118A mutation, suggesting that Cdc24p may interact with Cdc42p through its effector domain. The cdc42 G12V mutant phenotypes were suppressed by the intragenic T35A and K183-187Q mutations and in skm1⌬ and cla4⌬ cells but not ste20⌬ cells, suggesting that the mechanism of cdc42 G12V action is through the Skm1p and Cla4p protein kinases at the plasma membrane. Two intragenic suppressors of cdc42 G12V were also identified that displayed a dominant negative phenotype at 16°C, which was not suppressed by overexpression of Cdc24p, suggesting an alternate mechanism of action for these dominant negative mutations.The establishment of cell polarity is crucial for the control of many cellular and developmental processes, such as the generation of cell shape, the intracellular movement of organelles, and the secretion and deposition of new cell surface constituents (1). Polarized growth in the yeast Saccharomyces cerevisiae occurs in response to both internal and external signals, resulting in different morphological structures (2-5). The mechanics of cell polarity initiation during the mitotic cell cycle can be divided into three sequential phases: (i) nonrandom bud site selection; (ii) organization of proteins at the bud site; and (iii) bud emergence and polarized growth. Genetic and biochemical studies have identified over 25 proteins, including several GTPases and components of the actin cytoskeleton, that are involved in the regulation of the cell polarity pathway in S. cerevisiae (1, 6, 7).At least six members of the Ras superfamily of GTPases (Rsr1p/Bud1p, Cdc42p, Rho1p, Rho2p, Rho3p, and Rho4p) are involved in controlling cell polarity in S. cerevisiae. These proteins are active when in the GTP-bound state and inactive in the GDP-bound state (8, 9). The activity of these GTPases is controlled by regulatory proteins, such as guanine-nucleotide exchange factors, GTPase-activating proteins, and guanine-nucleotide dissociation inhibitors, as well as by the intracellular localization of the GTPase. Rsr1p/Bud1p is a member of the Ras subfamily and is responsible for bud site selection at one of the two cell poles, but it is not required for bud emergence or polari...
In the bacterium Escherichia coli, oxidized pyrimidines are removed by two DNA glycosylases, endonuclease III and endonuclease VIII (endo VIII), encoded by the nth and neigenes, respectively. Double mutants lacking both of these activities exhibit a high spontaneous mutation frequency, and here we show that all of the mutations observed in the double mutants were G:C→A:T transitions; no thymine mutations were found. These findings are in agreement with the preponderance of C→T transitions in the oxidative and spontaneous mutational databases. The major oxidized purine lesion in DNA, 7,8-dihydro-8-oxoguanine (8-oxoG), is processed by two DNA glycosylases, formamidopyrimidine DNA glycosylase (Fpg), which removes 8-oxoG opposite C, and MutY DNA glycosylase, which removes misincorporated A opposite 8-oxoG. The high spontaneous mutation frequency previously observed in fpg mutY double mutants was significantly enhanced by the addition of the neimutation, suggesting an overlap in the substrate specificities between endo VIII and Fpg/MutY. When the mutational specificity was examined, all of the mutations observed were G:C→T:A transversions, indicating that in the absence of Fpg and MutY, endo VIII serves as a backup activity to remove 8-oxoG. This was confirmed by showing that, indeed, endo VIII can recognize 8-oxoG in vitro.
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